The differentiation of IgM+ B cells into Ab secreting cells occurs in response to an array of signals provided by activated CD4+ T cells. A critical molecule in this regard is the CD40 ligand (CD40L, CD154), which is expressed on activated T cells and through its interaction with CD40 on B cells, drives the proliferation and differentiation of antigen-activated B cells. Although the relationship between proximal CD40 signaling events and downstream functional effects is not fully elucidated, it is known that this multi-step process requires the activation of a myriad of signal transduction pathways triggered by the binding of TRAF adapter molecules and the relocation of signaling complexes into the lipid insoluble fractions. The association of TRAF molecules with CD40 have been extensively characterized, however, the very early events that initiate the firing of responding pathways and how these signals are integrated to effect B cell activation are not fully understood. Recent work suggests that CD40 signaling acts more like a rheostat rather than an on-off switch with respect to the initiation of downstream functions. We have developed a unique B cell line from an atypical hyper-IgM patient (pt1) using a mini-EBV viral vector, which expresses the latent membrane protein (LMP)1 under the control of an inducible tetracycline promoter. Like the primary B cells from pt1, these conditionally transformed cells show defective CD40 signaling in the absence of tetracycline. Recently, we have mapped the pt1 defect to a proximal event in CD40 activation;namely the recruitment of CD40 into lipid rafts. Also, in the presence of LMP1 there is normal proliferation but a higher level of apoptosis suggesting that LMP1 is not capable of completely complementing the pt1 defect. The goals of this proposal are to use this unique reagent to define the very early signaling events in CD40 activation and to understand the relationship of these early signaling events to functional outcomes. Specifically, we propose to 1) identify the early events leading to CD40 signaling with a focus on the role CD40 phosphorylation plays in regulating this process;2) analyze the localization of CD40 in the membrane of Pt1 and control cells upon signaling and identify interacting molecules at different times after activation;and 3) characterize the apoptosis and defective c-Rel expression that occur in Pt1 B cells with LMP1 signaling. Completion of these studies should lead to an increased understanding of how B cells utilize diverse signaling cascades to induce CD40-mediated B cell activation. Project Description